The objective of this research program is to characterize the mechanisms of insulin induction of mRNA synthesis at the molecular level. To approach that problem, this project will utilize three specific cloned cDNA probes against three insulin-inducible RNA species. Two of these probes have already been cloned and selected for use by Northern analysis. The are directed against inducible RNA species of 20S and 28S. The 20S RNA species directs the synthesis of a discrete 50K protein. The third probe will be cloned as part of this program. This probe will be directed against the important metabolic enzyme pyruvate kinase. Pyruvate kinase plays a pivotal role in glycolysis, and the modulation of the expression of the hepatic isozyme is a critical and significant factor in redirectly hepatic metabolic response during dietary adaptations, metabolic disease states such as diabetes, or neoplasia. Information on the site of insulin action on the processing and turnover of each of the three mRNA species will be obtained using hybridization analysis of cytosolic, polysomal and nuclear RNA preparations. It is expected that insulin can act at the genomic level, and the isolation and characterization of each of the three genes coding for these RNAs will be undertaken as the first step in developing a hormonally responsive in vitro transcriptional system. These studies should greatly expand our understanding of peptide hormone action at the molecular level, specifically as it relates to insulin regulation of mRNA synthesis. Also, by characterizing three different RNA species, a unified or diversified theory of insulin action can be approached.